Biochemical sensor with thermoelastic probes

ABSTRACT

The thermoelastic response of certain materials to an incident electromagnetic excitation beam is highly sensitive to physical conditions existing at the surface of the material. Probe structures carrying probe materials are used as sensors in the analysis and investigation of biochemical molecules. Each probe structure is adapted to undergo a thermoelastic response when excited by temporally varying electromagnetic radiation, characteristics of the thermoelastic response being a function of physical properties of material binding to the surface of the probe structure. An electromagnetic excitation means directs electromagnetic energy at a selected one of the probe structures in order to elicit the excitation response. A detection means determines change in excitation response of the probe structures resulting from the binding of molecules thereto.

The present invention relates to a transducer sensor device, and inparticular to an array of such devices that can be used in the analysisof molecular structures, e.g. of bio-chemicals.

For the efficient analysis and investigation of biochemical molecularstructures, such as that which occurs during DNA sequencing, there is astrong demand for analytical tools that enable the use of hundreds oreven thousands of molecular probes substantially simultaneously.

One way of achieving this is to provide a substrate with a large numberof different probe molecules bound to its surface in an array. Eachprobe molecule is adapted to bind with a selected target molecule in asample under analysis. The sample is first provided with suitablefluorescent markers prior to exposure to the array of probe molecules.After exposure of the sample to the array, provided that the locationand identity of each different probe molecule in the array is known,analysis of the sample is possible using a confocal microscope toidentify array positions in which fluorescence indicates the presence ofa sample molecule bound to the respective probe molecule.

Typically the probe molecules are oligonucleotides and the sample underanalysis is a DNA sequence. Using such fluorescent techniques relativelyhigh probe densities are possible.

A disadvantage of such techniques is that the sample must be pre-treatedwith fluorescent markers to allow for fluorescence detection afterexposure to the probe array. A further disadvantage is that microscopeimaging systems can be costly and inconvenient for rapid analysis.

Surface plasmon resonance (SPR) is based on an optical phenomenon thatoccurs in a thin metal film at an optical interface under conditions oftotal internal reflection. Conventional SPR sensors use a prism ‘device’coated with a single thin metal layer. Any chemical adsorption to theouter surface of the metal layer or to an immobilised antibody or ligandon the outer surface of the metal layer leads to interfacial changes inthe refractive index of the film. By directing a light beam into theprism, it is possible to measure the reflected light as a function ofintensity and angle, to produce the well known SPR resonance spectrum.In a recent extension of this concept (U.S. Pat. No. 6,373,577) planarwaveguide elements coated with a thin metal film are organised as alinear array of elements in which SPR can be separately generated.

The present invention is directed to an improved system and apparatusfor analysing a sample using one, or an array, of probes that does notrequire the use of markers, that does not involve SPR, and that canreadily be implemented using low cost equipment. In contrast to SPR, thepresent invention is based on a high peak power beam of electromagneticradiation which temporarily illuminates a small region of athermoresponsive sensor, such as a thin metal layer. The incident angleof the beam is constant and the electromagnetic radiation induces athermal response that can be detected with an appropriate transducer.Chemical adsorption to the surface modulates the thermal response, whichin turn affects the electrical output of the transducer.

According to one aspect the present invention provides an apparatus fordetecting a variation in a probe, comprising:

-   -   a probe which is adapted to undergo one or more of a        thermoelastic, thermoelectric or thermomagnetic excitation        response when excited by temporally varying electromagnetic        radiation, the excitation response being a function of the        physical and/or chemical properties of the probe and/or of        material binding thereto;    -   a source of electromagnetic radiation;    -   means for directing the electromagnetic radiation at the probe;        and    -   a transducer adapted to determine the excitation response of the        probe.

The probe usually comprises a plurality of probe molecules (probematerial) carried on a separate surface, e.g. the surface of asubstrate. The substrate is preferably a thin film. However where theprobe material on its own possesses appropriate physical properties theprobe may be such material on its own.

We prefer the apparatus to comprise a plurality of probes, e.g. in theform of an array. Each probe may comprise probe material which isdifferent to that on other probes.

The probe material may consist of a collection of the same molecules orof a mixture of different molecules.

The surface of the probe may be a flat, e.g. a flat plate, but thesurface may also be curved, e.g. a segment of a sphere and such curvedsurfaces are included in the term “plate”.

The thermal excitation response may be thermoelectric, thermomagnetic orpreferably thermoelastic.

Preferably the source of electromagnetic radiation emits radiation inthe optical portion of the electromagnetic spectrum, and more preferablyis a laser. The source of electromagnetic radiation may be positioned sothat the radiation impinges directly on the probe material, but moreusually is positioned so that it first passes through a substratetransparent to the radiation before impinging on the probe material.

According to another aspect the invention provides a sensor, comprising:

-   -   a substrate, and    -   one or more probes on the surface of the substrate, the probes        being adapted to undergo one or more of a thermoelastic,        thermoelectric or thermomagnetic excitation response when        excited by temporally varying electromagnetic radiation, the        excitation response being a function of the physical and/or        chemical properties of the probe(s) and/or material binding        thereto.

The sensor is preferably in the form of a plate.

The substrate is preferably electromagnetically transparent, and may actas, or be part of, a transducer. The substrate is conveniently of such athickness that it has sufficient strength for ease of handling, and alsothat it will permit the desired amount of electromagnetic radiation topass through it. A convenient thickness is generally in the range 0.2 to1.0 mm.

Preferably the sensor includes:

-   -   a plurality of probes, e.g. thin film probe structures as        defined below, each of which is adapted to undergo a localised        electrical response when the probe is excited by temporally        varying electromagnetic radiation and of generating an        electrical output response corresponding thereto, the        characteristics of the electrical response being a function of        the physical or chemical properties of the probe and/or of        material binding thereto, and    -   a transducer for transmitting the electrical response.

Preferably different probe materials are bound to different probes. Whenthe probes comprise probe materials bound to a separate surface weprefer the surface to carry a layer, and preferably a thin layer, e.g.of silica, for receiving the respective probe materials and assistingthe probe materials to adhere to the surface. The layer may, forexample, be about 10 nm thick

In one aspect the present invention utilises the phenomenon that thethermoelastic, thermoelectric or thermomagnetic response of a probe canbe highly sensitive to physical and/or chemical conditions existing atthe surface of the probe. Indeed it has been shown that thethermoelastic (acoustic) response characteristics can vary substantiallyas a function of mass bound to a surface, surface stiffness, dielectricconstant, viscosity, surface free energy and other general surfaceproperties.

The present invention exploits this phenomenon by, for example,providing one or more independent locations (spots or dots) on asubstrate surface for which the thermal (e.g. thermoelastic) responsecan be assessed either qualitatively or quantitatively. When more thanone location is involved the response characteristics can be assessedseparately. The locations are preferably formed as an array of probes,the array preferably having a density of between 100 and 1,000,000locations per square centimetre, and more preferably between 100 and100,000 locations per square centimetre. Each location in the arrayeffectively provides an independently addressable probe. Each probe canbe excited to generate the thermal response by way of a beam ofelectromagnetic radiation, e.g. a laser beam.

Under an electromagnetic, e.g. laser, beam the probes absorb energy,e.g. optical energy, which is converted into thermal energy, which maybe vibrational or acoustic energy, referred to herein as a thermoelasticresponse. The periodic signal derived from a pulsed laser is ideal forlinking high performance detection circuits that are based onsynchronous detection thus providing a high signal to noise ratio. Shortnano or sub-nano second heating of the probe produces a localisedvolumetric expansion in the probe as a function of its density, thermalexpansion coefficient and reflection coefficient.

Where the probe has one or more molecules derived from the sample to beanalysed attached, e.g. chemically immobilised, bound or otherwise fixedthereto, the localised volumetric expansion will be modulated by theattached molecules thereby providing a differential thermoelasticexpansion. This modulation in the thermoelastic response is used in thepresent invention.

By means of this invention very small probes can be used and inconsequence attachment of even a small amount of sample to the probewill cause a relatively great change in the properties of the probe andthus provide a high degree of sensitivity.

Embodiments of the present invention will now be described by way ofexample with reference to the accompanying drawings, in which likenumerals denote like parts, and:

FIG. 1 is a schematic diagram illustrating the principles of the presentinvention;

FIG. 2 is a schematic diagram of a first embodiment of the inventionusing a laser based excitation system and an optical detection system;

FIG. 3 is a schematic diagram illustrating a further embodiment of theinvention using a laser based excitation system and a piezoelectricdetection system;

FIG. 4 is a schematic diagram illustrating a further embodiment of theinvention using a laser based excitation system and a thin filmtransducer detection system;

FIGS. 5A, 5B, 5C and 5D are schematic diagrams showing four differentexcitation and detection systems illustrating detection in both time andfrequency domains;

FIGS. 6A, 6B and 6C are illustrations of typical thermoelastic responsewave forms using the excitation and detection systems of FIG. 5C; and

FIGS. 7A, 7B and 7C illustrate the response of the system of FIG. 5C tovarious reactions.

With reference to FIG. 1, a transducer apparatus 1 for detectingspatially localised variations in material binding to a substrate isshown. A substrate 10, preferably formed from glass or similar material,has one or more thermoelastic sensing thin film structures 11 attachedto the top surface 12 of the substrate in a conveniently configuredarray. Each of the sensing structures 11 has probe material 13 attachedto the exposed upper surface of the thermoelastic thin film structures.The thin film structures will hereinafter be referred to as probestructures.

Preferably the probe structures 11 comprise dots or spots of anysuitable shape having a surface area of approximately 10⁻⁶ to 10⁻⁵square centimetres. In a preferred embodiment the probe materialcomprises an oligonucleotide adapted to bind with specific DNAfragments. Each probe structure, or group of probe structures, hasdifferent oligonucleotide probe materials bound thereto.

In the present embodiment, the substrate 10 is optically transparent,and positioned beneath it is an optical source 14, e.g. a Q-switchedlaser, for delivering an excitation signal to the probe structurethrough the thickness of the substrate. Together with the optical sourceis an optical detection system 15 for detecting modulations in opticalradiation reflected or returned from the probe structures. Theexcitation and detection systems are displaceable relative to thesubstrate, preferably in a plane substantially parallel to thesubstrate, and optionally also relative to each other, to enablescanning of the array.

A sample 16 comprising a plurality of fragments 17, e.g. DNA fragmentsin a buffer solution, is brought into contact with the top surface 12 ofthe substrate where specific fragments 17 bind with specific probematerial 13. The binding of the sample 16, 17 with the probe material 13of specific probe structures 11 results in a change in thecharacteristics of the thermoelastic response induced in the probestructures and this change is detected using the excitation anddetection systems 14, 15.

Excitation and Detection

With reference to FIG. 2 a preferred arrangement of excitation anddetection system will now be described.

A laser source 14 delivers an excitation beam 20 of suitable wavelength(e.g. 1056 nm) to a beam splitter 21. A first portion 20a of theexcitation beam is transmitted by the beam splitter 21 to the substrate10, and a second portion 20 b is dissipated at the detection system 15.The first portion 20 a of the excitation beam impinges on the substrate10, is transmitted through the substrate and directed onto a selectedprobe structure 11.

A detection beam 23, from a continuous, low powered laser source 22 isalso directed to the beam splitter 21. A first portion 23 a of thedetection beam is reflected by the beam splitter to the substrate 10where it is reflected from the probe structure and deflected to thedetection system 15 as an interference beam 23 b. A second portion 23 cof the detection beam is transmitted to the detection system directly asa reference beam. Interference between the two detection beam paths 23 band 23 c occurs and this interference is detected by the detectionsystem 15. The detection beam 23 b is preferably broader than the probestructure so that it can detect excitation over the whole of the probestructure.

In this exemplary embodiment the probe structure 11 preferably comprisesa metal film of thickness approximately 10 to 500 nm (and morepreferably 10 to 100 nm), and having a diameter of approximately 1 to100 μm. Alternatively the individual probe structures may be definedwithin a continuous film with the probe area effectively defined by theexcitation beam area.

The probe structure may be formed from any suitable metallic or othermaterials that provides the requisite thermoelastic properties, andwhich permits binding of suitable probe materials thereto, or chemicalmodification for attachment of suitable probe materials. Preferred forease of chemical attachment is gold, and for its thermoelasticproperties is aluminium. Other suitable materials include silver,titanium, copper, tungsten and polymeric materials.

A proportion of the excitation beam 20 a is absorbed by the probestructure 11 causing a thermoelastic volume change in the probestructure. This volume change results in one or more of a change in thethickness, area or position of the probe structure 11. For example, theexcitation beam produces longitudinal waves in the probe structure 11driven by localised heating of the metal.

In a preferred arrangement, the power density of excitation beam fallingonto the substrate is of the order of 3×10¹¹ W.m⁻² and this powerdensity yields a maximum strain on the probe structure of about 2%, ie.the width or thickness of the thermoelastic film increases by thisamount. Generally, the minimum power density required of the excitationbeam will depend upon the minimum thermoelastic response measurable bythe detection apparatus. In a present embodiment, this minimum powerdensity of excitation beam would be of the order of 3×10⁸ W.m⁻².

The interferometer formed from the combination of a. the beam 23 a thatis reflected off the probe structure 11 as beam 23 b, with b. thereference beam 23 c operates between the short duration pulses of theexcitation beam. The thermoelastic change in dimension of the probestructure results in a corresponding amplitude, phase and phase anglevariation at the photodiode detector 15. The position and expansion ofthe probe structure 11 is a function of the probe material bound thereto(and/or of any sample material which is attached to the probe material).

The result is that the amplitude, phase and phase angle of the responseto excitation measured at the photodiode 15 is directly related to thequantity of material bound to the probe structure 13. Similarly anystorage of energy in the probe structure results in vibrational, e.g.acoustic, resonance, that decays with time and, to a firstapproximation, is inversely proportional to the quantity of materialbound to-the probe structure 13, as will be shown later in FIG. 6.

In general terms the thermoelastic response of the probe structure dueto the electromagnetic, e.g. laser, excitation results in a change inhow the probe structure reflects light. Changes in the thermal responsewill occur as a function of any changes that occur to the physicaland/or chemical properties of the material bound to the surface of theprobe structure, which can be detected by the detection system 15. Ifthe excitation response is initially measured for the probe structurewith the probe material 13 bound thereto (calibration data), and is thenre-measured after exposure of the probe material to the sample 16(sample data) any changes to the physical and/or chemical properties ofthe material (e.g. any fragments 17) binding to the surface of the probestructure will be indicated, e.g. quantitatively, by the magnitude ofchange in the thermal response. For certain analytical purposes aqualitative response may be sufficient.

The data for sample 16 is acquired by the difference between thecalibration data and the sample data. Fast analogue to digitalconverters (for example, a transient recorder 25 such as a digitisingoscilloscope) translate this information into a series of digitalwaveforms for analysis by available software. Recording and storing datafrom the interactions of known probe materials on the probe structureswith known fragments in a sample enables the rapid identification ofsuch fragments in samples of unknown composition. An advantage of awideband waveform acquisition system, e.g. an oscilloscope, is that thetime domain signal is a fall record of the excitation from its initialmotion to its eventual relaxation. Where a less detailed emissionresponse is required it is possible to use a low frequency synchronousapproach, e.g. a lock-in amplifier which is simple in form and does notrequire radio frequency components.

Those skilled in the art will recognise that the thermoelastic responsemay be measured in a number of ways.

The excitation energy of the laser 14 may be in the form of a singlepulse (e.g. where only qualitative data is required), or a series ofpulses (e.g. where quantitative data is required). For each pulse theinduced stress response of the probe structure to the rising edge of theexcitation pulse may be analysed from the received signal and displayedon an oscilloscope (not shown).

Alternatively the Q-switching rate of the excitation laser may be usedto synchronise a lock-in amplifier in order to provide an enhancedsignal to noise ratio.

With reference to FIG. 3 an alternative embodiment of probe structure,excitation system and detection system will now be described. In thisarrangement each probe structure 30 is formed in a continuous thin film32 of electrically conductive, thermoelastic material on top of adielectric, optically transparent substrate 31. The thin film 32 canprovide an upper electrode of a piezoelectric transducer. Preferably theoptically transparent substrate is quartz.

In a preferred arrangement, a lower electrode 34, formed on the lowersurface of the substrate 31 is an optionally apertured entrant electrodewhich provides access for an excitation beam 20 to the probe structure30. The entrant electrode comprises a film of suitable electricallyconductive material to provide a ground plane and thereby reduceexternal electromagnetic interference. The size of the probe structure30 may therefore be effectively determined by the dimension of theexcitation beam 20. If the entrant electrode is formed from an opticallytransparent electrically conducting material, e.g. an indium/tin oxidecomposition, then no aperture need be formed.

The electromagnetic excitation beam 20 in this instance comprises anoptical beam generated by a Q-switched laser 14 and is preferably of theorder of 1.0 to 100 [m wide. The probe structure 30 responds toincidence of the optical excitation beam by thermoelastic volume changestherein according to the intensity and location of the beam. Thisproduces a vibrational, e.g. an acoustic, response in the probestructure 30 which induces movement in the adjacent piezoelectricsubstrate 31 and thereby produces a signal current 35.

The result is that vibration induced in the probe structure 30 iscoupled into the substrate 31. A wide range of frequencies is generatedas a high frequency current.

It will be understood that in this embodiment the thermoelastic responseof the probe structure 30 due to laser excitation results in amodulation of the electronic properties of the probe structure, e.g. itwill drive the thermoelastic excitation response. Changes in thisthermoelastic response will occur as a function of any changes thatoccur to the physical and/or chemical properties of the material bindingto the surface of the probe structure, which can be detected by anelectrically based detection system, rather than an optically baseddetection system.

The frequency and amplitude of the acoustic wave in the probe structureand thus of the induced signal current, is a function of the physicaland/or chemical properties of material 33 bound to the surface of theprobe structure 30. Thus, if the mass or other physical and/or chemicalproperties of the material 33 bound to the surface of the probestructure 30 changes, a consequent change in the amplitude and frequencyof the current 35 is observed. As described below this can be monitoredon an oscilloscope. FIG. 6A shows the thermoelastic response signalsincluding the initial response when the excitation laser beam strikesthe probe structure. FIG. 6B indicates the decay of the stored acousticenergy in the probe structure, while FIG. 6C is the Fourier Transform ofthe received thermoelastic response signal indicating the variousfrequency components that are stimulated.

Changes that occur to the physical and/or chemical properties of thematerial bound to the probe structure can be detected by the detectionsystem 15. If the excitation response is initially measured for theprobe structure with the probe material 33 bound thereto, and is thenmeasured again after exposure of the probe material to the sample 16,any changes to the physical and/or chemical properties of the materialbinding to the surface of the probe structure will be indicated, e.g.quantitatively, by the magnitude of change in the excitation response.

With reference to FIG. 4 an alternative embodiment of the probestructure, excitation system and detection system will now be described.Like the arrangement of FIG. 3 this embodiment also generates anelectrical response to the excitation beam. In this arrangement theprobe structure 40 is formed on a thin optically transparent substrate10. The probe structure is a layered structure comprising an electrode40 a formed on the substrate, a transduction film 40 b formed thereon,and an adhesion coating 40c on top; the coating 40 c being such as tofacilitate binding of probe material 44 thereto.

The optical excitation beam 20, 20 a in this instance (and some otherinstances) may alternatively be directed from above the substrate, shownat 14 a. The excitation beam 20, 20 a is directed onto the probestructure 40, and the resulting thermoelastic, thermoelectric and/orthermomagnetic response of the transduction film 40 b generates adetectable electrical output via the electrode 40 a.

An infrared emission output by the probe structure can be detected by atransduction film 40 b that is a pyroelectric film. An acoustic emissionby the probe structure can be detected by the electrode, e.g. a metalfilm electrode, 40 a. In the case of a thermomagnetic excitationresponse the transduction film 40 b could be a magnetoresistive layerthat changes its resistance according to the magnetic field emissionfrom the probe structure.

In common with the system of FIG. 2 the infrared, acoustic, magnetic orcharge emission response of the probe structures vanes as a function ofthe physical and/or chemical properties of the material binding to thesurface of the probe structure. If the mass bound to the surface of theprobe structure increases a changed, usually a greater, emissionresponse is observed.

In the preferred embodiments, the transducer element provides for adirect electrical pickup of signal current therefrom. In an alternativeembodiment the signal pickup could be remote, e.g. by electromagneticinduction. For example the piezoelectric transducer can provide anelectromagnetic signal that can be detected remotely by suitableantennae according to known principles.

Alternatively, charge emission can be detected by the electro-opticaleffect (Kerr, Pockels or Faraday) of an appropriate transduction film,which will change its refractive index and would be detectable as anoptical signal according to known techniques.

With reference to FIGS. 5A to 5D four alternative arrangements ofexcitation and detection systems are described.

FIG. 5A shows a digital oscilloscope detection system. This is thepreferred system for the probe structures of FIG. 2 as it can trackprecise dimensional or positional changes in the probe structure. Theexcitation beam 20 is generated using a Q-switched laser 14 producingeither single emissions or being self-modulated at frequencies ofseveral, e.g. 10 to 200, kHz according to well known techniques.

The detection (interference) beam 23 b is directed to an opticaldetection system 15 comprising a photodiode 52, a preamplifier 53 and adigitising oscilloscope 54 which is triggered by an optical detector 61adjacent to the Q-switched laser and optically coupled thereto by a beamsplitter 60.

The excitation beam 20 can be modulated by signals of up to several kHz,e.g. 10 to 200 kHz. The probe structure 11 is exposed to the pulsedexcitation beam 20 and increases in volume, e.g. by between about 0.1%and 10%. This leads to a change in phase and intensity in the detectionbeam 23 b due to changes, e.g. interference, in the optical path, and inthe beam area, this latter as a result of a change in the size of theprobe structure. Typically, the acceleration of the lateral movement inthe probe structure 11 is detectable from intensity changes in thedetection beam 23 b and corresponds to a mass change of material boundto the probe structure. Mass changes in the range 10⁻¹⁴ to 10⁻¹⁰ may bedetectable in preferred embodiments. In addition reflection at theboundaries of the probe structure leads to a characteristic resonantdecay that typically has a frequency of between 20 MHz and 200 MHz and adecay constant of between 10² and 10⁸ s⁻¹. Any change of the materialbound to the probe structure 11 changes the form of the decay.

It will be understood that the digitising oscilloscope may communicatethe results with a suitable automated digital storage and processingsystem (not shown) for rapid assessment of many excitation responsesfrom different probe structures on the substrate.

FIG. 5B shows a lock-in amplifier detection system 58 also suited to theprobe structures of FIG. 2. Most components are similar to thosedescribed with reference to FIG. 5A as indicated by the common referencenumerals, with the addition of the digital filter 62, which acts toreduce noise. In this case however the repeating output of a Q-switchedlaser 14 is used as a 10 to 200 kHz reference signal. The filterfrequency is selected to the appropriate acoustic emission frequency ofthe probe structure in order to optimise the signal to noise ratio ofthe detection signal. The response frequency typically lies in the range1 to 2000 MHz and deviates by a maximum of 10% when fragments 17 areadsorbed onto the probe structure 11.

FIG. 5C shows an oscilloscope detection system 36 particularly adaptedto the probe structures of FIG. 3 for direct detection of thethermoelastic response from the output signal current 35. In this systemthe Q-switched laser 14 produces the trigger signal (at optical detector61) for the oscilloscope 36. The electrical detection signal (outputsignal 35) of the probe structure 30 is applied to the oscilloscope viapreamplifier 53. The Fourier transform of the output signal produced bythe pulsed laser 14 is used to determine more detailed characteristicsof the fragments 17.

After adsorption of fragments 17 onto the probe material 13, changes inthe frequency and time decay can be observed, and these changes can beused to evaluate the fragments 17 adsorbed on the probe structure, andhence the sample 16.

FIG. 5D shows an alternative synchronisation detection system thatsubstantially filters the electrical emission signal 35 from thebackground noise using a digital filter 62. This eliminates noise fromunwanted frequency ranges and allows higher gain amplification. Thesystem coherently integrates the emission signal 35 from the transducer,averaging extraneous signals to zero.

In all the embodiments of FIGS. 5A, 5B, 5C and 5D typical signal voltageoutputs are in the range 10 μV to 100 μV and offer sufficientsensitivity to detect, for example probe structure mass changes of 10⁻¹⁴to 10⁻¹⁰ g.

An example of the electrical detection signal received from theelectrode by the oscilloscope is shown in FIG. 6. FIG. 6A illustratesthe excitation response of the probe structure to a pulsed excitationbeam. The slope indicated at 70 provides a measure of the thermoelasticacceleration of the probe structures. FIG. 6B illustrates the excitationresponse of the probe structure to a single pulse excitation. The decayprofile 71 provides a measure of the thermoelastic energy stored andreleased by the probe structure. FIG. 6C illustrates the frequencyspectrum 64 of the excitation response of the probe structure.

An exemplary characteristic response of the changes in thermoelasticresponse (vibration) of the probe structure before and after exposure tosucrose solutions is shown in FIG. 7A.

The effect of protein binding is shown in FIG. 7B and the effect ofhybridisation of polynucleotides is shown in FIG. 7C.

The results shown in FIG. 7 were obtained at 25° C. using a 100 nmaluminium thermoelastic layer on a quartz substrate. The sucrose wasdissolved in distilled water, and the protein and DNA solutions in PBSat pH 6.2. In FIGS. 7B and 7C Protein A and Poly C are used as the probematerials.

The upper curves are the time domain responses and the lower curves arethe frequency domain responses. Significant changes in response areobserved following these interface reactions.

Sample Delivery

Application of the sample to the probe structures may be achieved in anumber of different ways. For DNA analysis the DNA sample may beextracted from whole blood. Separation of the DNA is carried out bydielectrophoretic field, which transports the cells to a contactelectrode. An AC signal from 1 to 10 MHz source is applied to thecontact electrode to provide a transport force. This removes the needfor centrifuging the sample to separate the cells. With the cells at theelectrode a voltage pulse is applied to lyse the cells breaking throughthe membrane and releasing the cell contents. Excision enzymes are usedto cleave the genomic DNA strands to make sequenceable lengths similarto the length of an oligonucleotide on the probe structure. Temperaturesabove the annealing temperature-of the DNA are used to separate doublestrands and provide single strands for analysis. Exposure of the sampleto the array of probe structures 11 may occur in a single step,especially for small array areas. We also contemplate multiple stepexposure, e.g. by pipetting the sample onto each probe structure.

Coupling Chemistries

Probe materials 13, 33, 44, such as nucleic acids can be attached to theprobe structures 11 of the acoustic transducer arrays using varioussuitable chemistries, of which the following is a non-exhaustive list ofpossibilities. The coupling chemistries are indicated for theirpreferred substrate type.

1. Avidin or streptavidin can be adsorbed to a gold surface, followed byoligonucleotides labelled with a biotin moiety which then bindsirreversibly.

2. Amino-functionalised oligomers (3′ and 5′) can be attached to asilanised glass or silicon surface using glutaraldehyde.

3. Alkyl thiols can be attached to oligonucleotides and DNA. Thesethiols then assemble on a gold surface as an ordered monolayer film.

4. Carboxyl-modified surfaces of crystalline silicon will attach tothiol modified DNA by means of electrostatic adsorption of polylysineand a heterobifunctional cross-linker.

5. Aldehyde modified DNA oligonucleotides can be attached to a dextranacrylamide copolymer layer on glass, gold and silicon surfaces.

6. Alkoxysilanes such as aminopropyltriethoxysilane (APTES) are used toform a stable cross-linked film which is treated with succinic anhydrideto modify the amino group to a carboxylic acid moiety. An amino acidlinked nucleic acid will then bind via carbodiimide coupling.

7. 3-mercaptopropyltrimethoxysilane (MPS) can be used to attach thiolmodified DNA.

8. Glycidoxypropyl-triethoxysilane (GOPS) will also attach a thiolmodified DNA with a greater distance between the nucleic acid and thesurface of the probe structure or substrate.

9. DNA/nucleic acid can also be conjugated to a silane for directattachment to the probe structure surface.

10. Thiols attach the DNA to gold surfaces and silanes to silicasurfaces.

The techniques and apparatus described above offer very considerableadvantages in terms of reduced cost and complexity of analysisapparatus. Well known thin film lithographic or robotic spottingtechniques can be used to form the high density arrays of probestructures, particularly on rotatable discs. Existing compact discread/write technology can be used to provide the laser based excitationsystems and disc access mechanisms for positioning the laser withrespect to a rotating substrate. In such a system a drive means isprovided for rotating the disc relative to an axis and an indexing meansvaries the position of the electromagnetic excitation and detectionsystem relative to said axis, typically in a radial direction.

As a result the analysis apparatus can be made fully portable, beingonly a few kilograms in weight owing to the nature of the laser acoustictransducer. The analysis apparatus can be made largely or fullyautomatic designed for use by non-expert personnel, and does not requirecomplex chemical protocols. This provides for highly reliable analysis.No special environment for use is required (e.g. Light- or sound-free),unlike fluorescent techniques, and the apparatus is found to besubstantially noise free, being non-responsive to dust and opticalcontamination. Only material that is bound to the probe structures isdetected.

The probe structure transducers formed on the substrates have been foundto be sufficiently sensitive to enable detection of binding between DNAstrands and single base-pair differences. The acoustic frequencies usedcan be adjusted to obtain further increases in sensitivity.

The probe structure transducer elements can be formed from any suitablematerial, particularly gold, silver, aluminium, copper or tungsten, byevaporation or sputtering with photolithographic patterning techniqueswell known in the semiconductor industry, to define the array.

The substrate can be formed from any suitable material, e.g. soda glass,BK7 glass, borosilicate glass, sapphire, silica glass (vitreosil),crystalline quartz or plastics materials such as polystyrene,polycarbonate or polyethylene.

Some applications of the systems described herein are in largemolecule/small molecule interactions, large molecule/large moleculeinteractions, gaseous/solid interactions, genotyping, DNA sequencing andcell expression analysis.

Those skilled in the art will recognise that other embodiments notdescribed above are within the scope of the appended claims.

1. An apparatus for detecting a variation in a probe, comprising: aprobe which is adapted to undergo one or more of a thermoelastic,thermoelectric or thermomagnetic excitation response when excited bytemporally varying electromagnetic radiation, the excitation responsebeing a function of the physical and/or chemical properties of the probeand/or of material binding thereto; a source of electromagneticradiation; means for directing the electromagnetic radiation at theprobe; and a transducer adapted to determine the excitation response ofthe probe.
 2. The apparatus of claim 1 in which each probe comprises aprobe structure having a substrate surface onto which is bound probematerial.
 3. The apparatus of claim 2 in which the substrate ispreferably a thin film.
 4. The apparatus of claim 1 further comprising aplurality of probes.
 5. The apparatus of claim 4 in which the pluralityof probes are formed in an array.
 6. The apparatus of claim 4 in whicheach probe comprises probe material which is different to that on otherprobes.
 7. The apparatus of claim 2 in which the probe materialcomprises molecules of one type.
 8. The apparatus of claim 2 in whichthe probe material comprises a mixture of different molecules.
 9. Theapparatus of claim 1 in which the surface of the probe is planar. 10.The apparatus of claim 1 in which the surface of the probe is curved.11. The apparatus of claim 1 in which the source of electromagneticradiation emits radiation in the optical portion of the electromagneticspectrum.
 12. The apparatus of claim 1 in which the source ofelectromagnetic radiation is a laser.
 13. The apparatus of claim 1 inwhich the source of electromagnetic radiation is positioned so that theradiation impinges directly on the probe material.
 14. The apparatus ofclaim 1 in which the source of electromagnetic radiation is positionedso that it first passes through a substrate transparent to the radiationbefore impinging on the probe material.
 15. A sensor, comprising: asubstrate, and one or more probes on the surface of the substrate, theprobes being adapted to undergo one or more of a thermoelastic,thermoelectric or thermomagnetic excitation response when excited bytemporally varying electromagnetic radiation, the excitation responsebeing a function of the physical and/or chemical properties of theprobe(s) and/or material binding thereto.
 16. The sensor of claim 15 inthe form of a plate.
 17. The sensor of claim 15 in which the substrateis electromagnetically transparent.
 18. The sensor of claim 15 in whichthe substrate acts as, or is part of, a transducer.
 19. The sensor ofclaim 15 in which the substrate is of such a thickness that it hassufficient strength for ease of handling, and also that it will permitthe desired amount of electromagnetic radiation to pass through it. 20.The sensor of claim 19 in which the substrate has a thickness in therange 0.2 to 1.0 mm.
 21. The sensor of claim 15 further including: aplurality of probes each of which is adapted to undergo a localisedelectrical response when the probe is excited by temporally varyingelectromagnetic radiation and of generating an electrical outputresponse corresponding thereto, the characteristics of the electricalresponse being a function of the physical or chemical properties of theprobe and/or of material binding thereto, and a transducer fortransmitting the electrical response.
 22. The sensor of claim 21 inwhich different probe materials are bound to different probes.
 23. Atransducer apparatus for detecting spatially localised variations inmaterial binding to the surface of a plate, comprising: a substrate; aplurality of thin film probe structures on a surface of the substrate,each probe structure being adapted to undergo one or more of a localisedthermoelastic, thermoelectric or thermomagnetic excitation response whenexcited by temporally varying electromagnetic radiation, characteristicsof the excitation response being a function of physical and/or chemicalproperties of material binding to the surface of the probe structure;electromagnetic excitation means for directing electromagnetic energy ata selected one of the probe structures in order to elicit the excitationresponse; and detection means for determining the excitation response ofthe probe structures.
 24. The apparatus of claim 23 in which theelectromagnetic excitation means emits said temporally varyingelectromagnetic radiation in the optical spectrum.
 25. The apparatus ofclaim 24 in which the substrate is formed from an optically transparentmedium, and in which the electromagnetic excitation means is adapted todirect said electromagnetic energy to a lower surface of the probestructure via the substrate.
 26. The apparatus of claim 25 in which theprobe structures are each adapted to absorb said electromagneticradiation to thereby generate a thermoelastic excitation response in theform of a volume change within the structure, and in which the detectionmeans comprises means for detecting said volume change in said probestructure.
 27. The apparatus of claim 26 in which the probe structureseach comprise a thin film metal spot.
 28. The apparatus of claim 26 inwhich the detection means comprises means for receiving reflectedelectromagnetic energy from the selected probe structure.
 29. Theapparatus of claim 25 in which the probe structures are each adapted toabsorb said electromagnetic radiation to thereby generate athermoelastic response in the form of a lateral displacement of thestructure, and in which the detection means comprises means fordetecting said lateral displacement of the probe structure.
 30. Theapparatus of claim 29 in which the probe structures each comprise a thinfilm dielectric material spot.
 31. The apparatus of claim 23 in whichthe probe structures include a transducer element for generating anelectrical output signal representative of a thermoelastic response ofsaid probe structures.
 32. The apparatus of claim 23 in which the probestructures include a transducer element adapted to provide athermoelectric excitation response to said temporally varyingelectromagnetic radiation, and in which the detection means comprisesmeans for detecting said thermoelectric excitation response.
 33. Theapparatus of claim 23 in which the probe structures include a transducerelement adapted to provide a thermomagnetic excitation response to saidtemporally varying electromagnetic radiation, and in which the detectionmeans comprises means for detecting said thermomagnetic excitationresponse.
 34. The apparatus of claim 23 in which the electromagneticexcitation means comprises a laser adapted to irradiate selected ones ofthe probe structures with pulsed or continuous wave electromagneticradiation.
 35. The apparatus of claim 23 in which the detection meanscomprises an optical interferometer for receiving a reference beam froman optical source, and an interference beam reflected from the probestructure.
 36. The apparatus of claim 23 in which the detection meansincludes a transient recorder or digitising oscilloscope for determiningan amplitude and phase variation in thermoelastic response signalsreceived from the probe structures.
 37. The apparatus of claim 23 inwhich the electromagnetic excitation means and the detection meansinclude means for detecting a change in resonant frequency of a selectedprobe structure.
 38. The apparatus of claim 23 in which each probestructure includes an entrant electrode adapted to provide a groundplane to a lower surface of the substrate.
 39. The apparatus of claim 23further including a molecular probe material bound to an exposed surfaceof the probe structure.
 40. The apparatus of claim 23 in which thesubstrate comprises a disc, and further including: drive means forrotating said disc relative to an axis; indexing means for varying theposition of said electromagnetic excitation means and said detectionmeans relative to said axis.
 41. A sensor plate comprising: an opticallytransparent substrate; and a plurality of thin film probe structures ona surface of the substrate, each probe structure being adapted toundergo one or more of a localised thermoelastic, thermoelectric orthermomagnetic excitation response when excited by temporally varyingelectromagnetic radiation, characteristics of the excitation responsebeing a function of physical and/or chemical properties of materialbinding to the surface of the probe structure.
 42. The sensor plate ofclaim 41 further including: a plurality of different molecular probematerials respectively bound to the exposed surfaces of a plurality ofthe probe structures.
 43. The sensor plate of claim 41 in which thesubstrate comprises silica.
 44. The sensor plate of claim 41 in whichthe probe structures each comprise a thin film metal spot.
 45. Thesensor plate of claim 41 in which the probe structures each comprise athin film dielectric spot.
 46. The sensor plate of claim 41 in whicheach probe structure further includes a transducer element forgenerating an electrical output signal representative of thethermoelastic response of said probe structure.
 47. The sensor plate ofclaim 41 in which each probe structure comprises a transducer elementadapted to provide a thermoelectric excitation response to saidtemporally varying electromagnetic radiation.
 48. The sensor plate ofclaim 47 further including an electrode for transmitting thethermoelectric excitation response to a detector.
 49. A sensor platecomprising: a substrate; a plurality of thin film probe structures on asurface of the substrate; each probe structure comprising a transductionfilm adapted to undergo one or more of a localised thermoelectric orthermomagnetic excitation response when the probe structure is excitedby temporally varying electromagnetic radiation and generating anelectrical output response corresponding thereto, characteristics of theelectrical output response generated being a function of physical and/orchemical properties of material binding to a surface of the probestructure; and an electrode for transmitting the electrical outputresponse.
 50. The sensor plate of claim 49 further comprising: aplurality of different molecular probe materials respectively bound to aplurality of said adjacent surfaces on the second face of the substrate.51. The sensor plate of claim 49 in which the probe structures furtherinclude a passivation layer over the transduction film for receiving therespective probe material binding to the surface of the probe structure.52. The sensor plate of claim 49 in which the substrate comprisessilica.
 53. The sensor plate of claim 49 in which the probe structurescomprise a magnetic material.
 54. The sensor plate of claim 41 in whichthe probe structures are arranged in a series of generally circular orhelical arrays on a circular disc substrate.
 55. A method of using atransducer apparatus according to claim 23 comprising the steps of:providing a plurality of probe materials respectively attached to aplurality of probe structures; exposing the probe structures to a samplematerial to permit binding of material to the surface of the probestructure; using the electromagnetic excitation means to directelectromagnetic energy at the probe structures; and detecting changes inexcitation response of each probe structure by comparing its excitationresponse with and without exposure to the sample material. 56.(canceled)
 57. (canceled)